Source-to-tap investigation of the occurrence of nontuberculous mycobacteria in a full-scale chloraminated drinking water system.

Nontuberculous mycobacteria (NTM) in drinking water are a significant public health concern. However, an incomplete understanding of the factors that influence the occurrence of NTM in drinking water limits our ability to characterize risk and prevent infection. This study sought to evaluate the influence of season and water treatment, distribution, and stagnation on NTM in drinking water. Samples were collected source-to-tap in a full-scale, chloraminated drinking water system approximately monthly from December 2019 to November 2020. NTM were characterized using culture-dependent (plate culture with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry [MALDI-TOF MS] isolate analysis) and culture-independent methods (quantitative PCR and genome-resolved metagenomics). Sampling locations included source waters, three locations within the treatment plant, and five buildings receiving water from the distribution system. Building plumbing samples consisted of first draw, 5-min flush, and full flush cold-water samples. As the study took place during the COVID-19 pandemic, the influence of reduced water usage in three of the five buildings was also investigated. The highest NTM densities source-to-tap were found in the summer first draw building water samples (107 gene copies/L), which also had the lowest monochloramine concentrations. Flushing was found to be effective for reducing NTM and restoring disinfectant residuals, though flush times necessary to improve water quality varied by building. Clinically relevant NTM species, including Mycobacterium avium, were recovered via plate culture, with increased occurrence observed in buildings with higher water age. Four of five NTM metagenome-assembled genomes were identified to the species level and matched identified isolates.IMPORTANCENTM infections are increasing in prevalence, difficult to treat, and associated with high morbidity and mortality rates. Our lack of understanding of the factors that influence NTM occurrence in drinking water limits our ability to prevent infections, accurately characterize risk, and focus remediation efforts. In this study, we comprehensively evaluated NTM in a full-scale drinking water system, showing that various steps in treatment and distribution influence NTM presence. Stagnant building water contained the highest NTM densities source-to-tap and was associated with low disinfectant residuals. We illustrated the differences in NTM detection and characterization obtained from culture-based and culture-independent methods, highlighting the complementarity between these approaches. We demonstrated that focusing NTM mitigation efforts in building plumbing systems, which have the highest NTM densities source-to-tap, has potential for immediate positive effects. We also identified steps during treatment that increase NTM levels, which provides beneficial information for utilities seeking to reduce NTM in finished water.

Genomic Analyses of Longitudinal Mycobacterium abscessus Isolates in a Multicenter Cohort Reveal Parallel Signatures of In-Host Adaptation.

BACKGROUND: Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and an increasingly frequent cause of opportunistic infections. Mycobacterium abscessus complex (MABC) is one of the major NTM lung pathogens that disproportionately colonize and infect the lungs of individuals with cystic fibrosis (CF). MABC infection can persist for years, and antimicrobial treatment is frequently ineffective. METHODS: We sequenced the genomes of 175 isolates longitudinally collected from 30 patients with MABC lung infection. We contextualized our cohort amidst the broader MABC phylogeny and investigated genes undergoing parallel adaptation across patients. Finally, we tested the phenotypic consequences of parallel mutations by conducting antimicrobial resistance and mercury-resistance assays. RESULTS: We identified highly related isolate pairs across hospital centers with low likelihood of transmission. We further annotated nonrandom parallel mutations in 22 genes and demonstrated altered macrolide susceptibility co-occurring with a nonsynonymous whiB1 mutation. Finally, we highlighted a 23-kb mercury-resistance plasmid whose loss during chronic infection conferred phenotypic susceptibility to organic and nonorganic mercury compounds. CONCLUSIONS: We characterized parallel genomic processes through which MABC is adapting to promote survival within the host. The within-lineage polymorphisms we observed have phenotypic effects, potentially benefiting fitness in the host at the putative detriment of environmental survival.

Testing the effects of combining azithromycin with inhaled tobramycin for P. aeruginosa in cystic fibrosis: a randomised, controlled clinical trial.

RATIONALE: Inhaled tobramycin and oral azithromycin are common chronic therapies in people with cystic fibrosis and Pseudomonas aeruginosa airway infection. Some studies have shown that azithromycin can reduce the ability of tobramycin to kill P. aeruginosa. This trial was done to test the effects of combining azithromycin with inhaled tobramycin on clinical and microbiological outcomes in people already using inhaled tobramycin. We theorised that those randomised to placebo (no azithromycin) would have greater improvement in forced expiratory volume in one second (FEV1) and greater reduction in P. aeruginosa sputum in response to tobramycin. METHODS: A 6-week prospective, randomised, placebo-controlled, double-blind trial testing oral azithromycin versus placebo combined with clinically prescribed inhaled tobramycin in individuals with cystic fibrosis and P. aeruginosa airway infection. RESULTS: Over a 6-week period, including 4 weeks of inhaled tobramycin, the relative change in FEV1 did not statistically significantly differ between groups (azithromycin (n=56) minus placebo (n=52) difference: 3.44%; 95% CI: -0.48 to 7.35; p=0.085). Differences in secondary clinical outcomes, including patient-reported symptom scores, weight and need for additional antibiotics, did not significantly differ. Among the 29 azithromycin and 35 placebo participants providing paired sputum samples, the 6-week change in P. aeruginosa density differed in favour of the placebo group (difference: 0.75 log10 CFU/mL; 95% CI: 0.03 to 1.47; p=0.043). CONCLUSIONS: Despite having greater reduction in P. aeruginosa density in participants able to provide sputum samples, participants randomised to placebo with inhaled tobramycin did not experience significantly greater improvements in lung function or other clinical outcomes compared with those randomised to azithromycin with tobramycin.

Liquid Chromatography Mass Spectrometry Detection of Antibiotic Agents in Sputum from Persons with Cystic Fibrosis.

Antibiotic therapy is expected to impact host microbial communities considerably, yet many studies focused on microbiome and health are often confounded by limited information about antibiotic exposure. Given that antibiotics have diverse pharmacokinetic and antimicrobial properties, investigating the type and concentration of these agents in specific host specimens would provide much needed insight into their impact on the microbes therein. Here, we developed liquid chromatography mass spectrometry (LC-MS) methods to detect 18 antibiotic agents in sputum from persons with cystic fibrosis. Antibiotic spike-in control samples were used to compare three liquid extraction methods on the Waters Acquity Quattro Premier XE. Extraction with dithiothreitol captured the most antibiotics and was used to detect antibiotics in sputum samples from 11 people with cystic fibrosis, with results being compared to the individuals' self-reported antibiotic use. For the sputum samples, two LC-MS assays were used; the Quattro Premier detected nanomolar or micromolar concentrations of 16 antibiotics, whereas the Xevo TQ-XS detected all 18 antibiotics, most at subnanomolar levels. In 45% of tested sputum samples (71/158), at least one antibiotic that was not reported by the subject was detected by both LC-MS methods, a discordance largely explained by the thrice weekly administration and long half-life of azithromycin. For ∼37% of samples, antibiotics reported as taken by the individual were not detected by either instrument. Our results provide an approach for detecting a variety of antibiotics at the site of infection, thereby providing a means to include antibiotic usage data into microbiome studies.

In Vitro Activities of ß-Lactam-ß-Lactamase Inhibitor Antimicrobial Agents against Cystic Fibrosis Respiratory Pathogens.

We tested the in vitro activities of ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam, piperacillin-tazobactam, and 11 other antimicrobial agents against 420 Burkholderia, Achromobacter, Stenotrophomonas, and Pandoraea strains, 89% of which were cultured from respiratory specimens from persons with cystic fibrosis. Among the ß-lactam-ß-lactamase inhibitor agents, meropenem-vaborbactam had the greatest activity against Burkholderia and Achromobacter, including multidrug-resistant and extensively-drug-resistant strains. None of the newer ß-lactam-ß-lactamase combination drugs showed increased activity compared to that of the older agents against Stenotrophomonas maltophilia or Pandoraea spp.

Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.

Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.

Microbial community organization designates distinct pulmonary exacerbation types and predicts treatment outcome in cystic fibrosis.

Polymicrobial infection of the airways is a hallmark of obstructive lung diseases such as cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Pulmonary exacerbations (PEx) in these conditions are associated with accelerated lung function decline and higher mortality rates. An understanding of the microbial underpinnings of PEx is challenged by high inter-patient variability in airway microbial community profiles. We analyzed bacterial communities in 880 CF sputum samples and developed microbiome descriptors to model community reorganization prior to and during 18 PEx. We identified two microbial dysbiosis regimes with opposing ecology and dynamics. Pathogen-governed PEx showed hierarchical community reorganization and reduced diversity, whereas anaerobic bloom PEx displayed stochasticity and increased diversity. A simulation of antimicrobial treatment predicted better efficacy for hierarchically organized communities. This link between PEx type, microbiome organization, and treatment success advances the development of personalized clinical management in CF and, potentially, other obstructive lung diseases.

Microbial community organization designates distinct pulmonary exacerbation types and predicts treatment outcome in cystic fibrosis.

Polymicrobial infection of the airways is a hallmark of obstructive lung diseases such as cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Pulmonary exacerbations (PEx) in these conditions are associated with accelerated lung function decline and higher mortality rates. Understanding PEx ecology is challenged by high inter-patient variability in airway microbial community profiles. We analyze bacterial communities in 880 CF sputum samples collected during an observational prospective cohort study and develop microbiome descriptors to model community reorganization prior to and during 18 PEx. We identify two microbial dysbiosis regimes with opposing ecology and dynamics. Pathogen-governed PEx show hierarchical community reorganization and reduced diversity, whereas anaerobic bloom PEx display stochasticity and increased diversity. A simulation of antimicrobial treatment predicts better efficacy for hierarchically organized communities. This link between PEx, microbiome organization, and treatment success advances the development of personalized clinical management in CF and, potentially, other obstructive lung diseases.

Impact of day-to-day variation in FEV1 on measures of change: A conceptual description.

Clinical trials often demonstrate treatment efficacy through change in forced expiratory volume in one second (FEV1), comparing single FEV1 measurements from post- versus pre-treatment timepoints. Day-to-day variation in measured FEV1 is common for reasons such as diurnal variation and intermittent health changes, relative to a stable, monthly average. This variation can alter estimation of associations between change in FEV1 and baseline in predictable ways, through a phenomenon called regression to the mean. We quantify and explain day-to-day variation in percent-predicted FEV1 (ppFEV1) from 4 previous trials, and we present a statistical, data-driven explanation for potential bias in ceiling and floor effects due to commonly observed amounts of variation. We recommend accounting for variation when assessing associations between baseline value and change in CF outcomes in single-arm trials, and we consider possible impact of variation on conventional standards for study eligibility.

Prevalence and Clinical Impact of Respiratory Viral Infections from the STOP2 Study of Cystic Fibrosis Pulmonary Exacerbations.

Rationale: Rates of viral respiratory infection (VRI) are similar in people with cystic fibrosis (CF) and the general population; however, the associations between VRI and CF pulmonary exacerbations (PEx) require further elucidation.Objectives: To determine VRI prevalence during CF PEx and evaluate associations between VRI, clinical presentation, and treatment response.Methods: The STOP2 (Standardized Treatment of Pulmonary Exacerbations II) study was a multicenter randomized trial to evaluate different durations of intravenous antibiotic therapy for PEx. In this ancillary study, participant sputum samples from up to three study visits were tested for respiratory viruses using multiplex polymerase chain reactions. Baselines and treatment-associated changes in mean lung function (percent predicted forced expiratory volume in 1 s), respiratory symptoms (Chronic Respiratory Infection Symptom Score), weight, and C-reactive protein were compared as a function of virus detection. Odds of PEx retreatment within 30 days and future PEx hazard were modeled by logistic and Cox proportional hazards regression, respectively.Results: A total of 1,254 sputum samples from 621 study participants were analyzed. One or more respiratory viruses were detected in sputum samples from 245 participants (39.5%). Virus-positive participants were more likely to be receiving CF transmembrane conductance regulator modulator therapy (45% vs. 34%) and/or chronic azithromycin therapy (54% vs. 44%) and more likely to have received treatment for nontuberculous Mycobacterium infection in the preceding 2 years (7% vs. 3%). At study visit 1, virus-positive participants were more symptomatic (mean Chronic Respiratory Infection Symptom Score, 53.8 vs. 51.1), had evidence of greater systemic inflammation (log10 C-reactive protein concentration, 1.32 log10 mg/L vs. 1.23 log10 mg/L), and had a greater drop in percent predicted forced expiratory volume in 1 second from the prior 6-month baseline (5.8 vs. 3.6). Virus positivity was associated with reduced risk of future PEx (hazard ratio, 0.82; 95% confidence interval, 0.69-0.99; P = 0.034) and longer median time to next PEx (255 d vs. 172 d; P = 0.021) compared with virus negativity.Conclusions: More than one-third of STOP2 participants treated for a PEx had a positive test result for a respiratory virus with more symptomatic initial presentation compared with virus-negative participants, but favorable long-term outcomes. More refined phenotyping of PEx, taking VRIs into account, may aid in optimizing personalized management of PEx.Clinical trial registered with www.clinicaltrials.gov (NCT02781610).

Effects of azithromycin in Pseudomonas aeruginosa burn wound infection.

BACKGROUND: Cutaneous thermal injuries (i.e., burns) remain a common form of debilitating trauma, and outcomes are often worsened by wound infection with environmental bacteria, chiefly Pseudomonas aeruginosa. MATERIALS AND METHODS: We tested the effects of early administration of a single dose of azithromycin, with or without subsequent antipseudomonal antibiotics, in a mouse model of standardized thermal injury infected with P aeruginosa via both wound site and systemic infection. We also tested the antimicrobial effects of these antibiotics alone or combined in comparative biofilm and planktonic cultures in vitro. RESULTS: In our model, early azithromycin administration significantly reduced wound and systemic infection without altering wound site or circulating neutrophil activity. The antimicrobial effect of azithromycin was additive with ciprofloxacin but significantly reduced the antimicrobial effect of tobramycin. This pattern was reproduced in biofilm cultures and not observed in planktonic cultures of P aeruginosa. CONCLUSION: These data suggest that early administration of azithromycin following burn-related trauma and infection may reduce P aeruginosa infection and potential interactions with other antibiotics should be considered when designing future studies.

Microbial community organization designates distinct pulmonary exacerbation types and predicts treatment outcome in cystic fibrosis.

Polymicrobial infection of the airways is a hallmark of obstructive lung diseases such as cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease (COPD). Intermittent pulmonary exacerbations (PEx) in these conditions are associated with lung function decline and higher mortality rates. An understanding of the microbial underpinnings of PEx is challenged by high inter-patient variability in airway microbial community profiles. We analyzed 880 near-daily CF sputum samples and developed non-standard microbiome descriptors to model community reorganization prior and during 18 PEx. We identified two communal microbial regimes with opposing ecology and dynamics. Whereas pathogen-governed dysbiosis showed hierarchical community organization and reduced diversity, anaerobic bloom dysbiosis displayed stochasticity and increased diversity. Microbiome organization modulated the relevance of pathogens and a simulation of antimicrobial treatment predicted better efficacy for hierarchically organized microbiota. This causal link between PEx, microbiome organization, and treatment success advances the development of personalized dysbiosis management in CF and, potentially, other obstructive lung diseases.

Airway bacterial community composition in persons with advanced cystic fibrosis lung disease.

BACKGROUND: The progression of lung disease in people with cystic fibrosis (pwCF) has been associated with a decrease in the diversity of airway bacterial communities. How often low diversity communities occur in advanced CF lung disease and how they may be associated with clinical outcomes is not clear, however. METHODS: We sequenced a region of the bacterial 16S ribosomal RNA gene to characterize bacterial communities in sputum from 190 pwCF with advanced lung disease (FEV1≤40% predicted), with particular attention to the prevalence and relative abundance of dominant genera. We evaluated relationships between community diversity and clinical outcomes. RESULTS: Although most of the 190 pwCF with advanced lung disease had airway bacterial communities characterized by low diversity with a dominant genus, a considerable minority (40%) did not. The absence of a dominant genus, presence of methicillin-susceptible Staphylococcus aureus, and greater bacterial richness positively correlated with lung function. Higher relative abundance of the dominant genus and greater antimicrobial use negatively correlated with lung function. PwCF with a low diversity community and dominant genus had reduced lung transplant-free survival compared to those without (median survival of 1.6 vs 2.9 years). CONCLUSIONS: A considerable proportion of pwCF with advanced lung disease do not have airway bacterial communities characterized by low diversity and a dominant genus and these individuals had better survival. An understanding of the antecedents of low diversity airway communities- and the impact these may have on lung disease trajectory - may provide avenues for improved management strategies.

Quantifying variation in home spirometry in people with cystic fibrosis during baseline health, and associations with clinical outcomes.

BACKGROUND: Home spirometry is increasingly used to monitor lung function in people with cystic fibrosis (pwCF). Although decreases in lung function in the setting of increased respiratory symptoms are consistent with a pulmonary exacerbation (PEx), the interpretation of home spirometry during asymptomatic periods of baseline health is unclear. The aims of this study were to determine the variation in home spirometry in pwCF during asymptomatic periods of baseline health and to identify associations between this variation and PEx. METHODS: Near-daily home spirometry measurements were obtained from a cohort of pwCF enrolled in a long-term study of the airway microbiome. Associations between the degree of variation in home spirometry and the time to next PEx were evaluated. RESULTS: Thirteen subjects (mean age of 29 years and mean percent predicted forced expiratory volume in one second [ppFEV1] of 60) provided a median of 204 spirometry readings taken during 40 periods of baseline health. The mean week-to-week within-subject level of variation in ppFEV1 was 15.2 ± 6.2%. The degree of variation in ppFEV1 during baseline health was not associated with time to PEx. CONCLUSIONS: Variation in ppFEV1 measured with near-daily home spirometry in pwCF during periods of baseline health exceeded the variation in ppFEV1 expected in clinic spirometry (based on ATS guidelines). The degree of variation in ppFEV1 during baseline health was not associated with time to PEx. These data are relevant for guiding interpretation of home spirometry.

The Impact of Highly Effective Modulator Therapy on Cystic Fibrosis Microbiology and Inflammation.

Highly effective cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator therapy (HEMT) corrects the underlying molecular defect causing CF disease. HEMT decreases symptom burden and improves clinical metrics and quality of life for most people with CF (PwCF) and eligible cftr mutations. Improvements in measures of pulmonary health suggest that restoration of function of defective CFTR anion channels by HEMT not only enhances airway mucociliary clearance, but also reduces chronic pulmonary infection and inflammation. This article reviews the evidence for how HEMT influences the dynamic and interdependent processes of infection and inflammation in the CF airway, and what questions remain unanswered.

The Elusive Role of Airway Infection in Cystic Fibrosis Exacerbation.

Cystic fibrosis (CF) pulmonary exacerbations (PEx) are clinical events that commonly result in increased treatment burden, decreased quality of life, and accelerated lung disease progression. CF PEx have historically been approached as though dealing with acute infections, and antibiotic treatments have been associated with improved outcomes. In this review, we discuss data supporting a causal role of CF airway infection in PEx as well studies that highlight our knowledge gaps in regard to PEx definitions, pathophysiology, and optimal treatment approaches. In the era of highly effective cystic fibrosis transmembrane conductance regulator modulator therapy, and the continually increasing health and longevity of persons with CF, a better understanding of PEx and further optimization of PEx antibiotic treatment approaches are needed.

Sputum Metabolites Associated with Nontuberculous Mycobacterial Infection in Cystic Fibrosis.

Nontuberculous mycobacterial (NTM) pulmonary infections in people with cystic fibrosis (CF) are associated with significant morbidity and mortality and are increasing in prevalence. Host risk factors for NTM infection in CF are largely unknown. We hypothesize that the airway microbiota represents a host risk factor for NTM infection. In this study, 69 sputum samples were collected from 59 people with CF; 42 samples from 32 subjects with NTM infection (14 samples collected before incident NTM infection and 28 samples collected following incident NTM infection) were compared to 27 samples from 27 subjects without NTM infection. Sputum samples were analyzed with 16S rRNA gene sequencing and metabolomics. A supervised classification and correlation analysis framework (sparse partial least-squares discriminant analysis [sPLS-DA]) was used to identify correlations between the microbial and metabolomic profiles of the NTM cases compared to the NTM-negative controls. Several metabolites significantly differed in the NTM cases compared to controls, including decreased levels of tryptophan-associated and branched-chain amino acid metabolites, while compounds involved in phospholipid metabolism displayed increased levels. When the metabolome and microbiome data were integrated by sPLS-DA, the models and component ordinations showed separation between the NTM and control samples. While this study could not determine if the observed differences in sputum metabolites between the cohorts reflect metabolic changes that occurred as a result of the NTM infection or metabolic features that contributed to NTM acquisition, it is hypothesis generating for future work to investigate host and bacterial community factors that may contribute to NTM infection risk in CF. IMPORTANCE Host risk factors for nontuberculous mycobacterial (NTM) infection in people with cystic fibrosis (CF) are largely unclear. The goal of this study was to help identify potential host and bacterial community risk factors for NTM infection in people with CF, using microbiome and metabolome data from CF sputum samples. The data obtained in this study identified several metabolic profile differences in sputum associated with NTM infection in CF, including 2-methylcitrate/homocitrate and selected ceramides. These findings represent potential risk factors and therapeutic targets for preventing and/or treating NTM infections in people with CF.

Changes in airway bacterial communities occur soon after initiation of antibiotic treatment of pulmonary exacerbations in cystic fibrosis.

Chronic polymicrobial airway infections are a hallmark of cystic fibrosis (CF) lung disease. Antibiotic therapy is a primary treatment of CF pulmonary exacerbations (PEx); however, the impact of episodic antibiotic treatment on airway bacterial communities has not been well described. We analyzed sputum samples from adults with CF obtained immediately before and during antibiotic treatment of PEx. Sequencing of the V4 region of the bacterial 16S ribosomal RNA gene was used to assess changes in bacterial community structure during antibiotic treatment. The peak impact of antibiotic treatment was observed by day four or five of treatment. These findings advance our understanding of bacterial community dynamics during antibiotic treatment of PEx and complement recent and ongoing studies evaluating the optimal duration of antibiotic therapy for PEx.